Thermal unfolding of proteins at high temperatures is caused by a strong increase of the entropy change which lowers Gibbs free energy change of the unfolding transition (ΔGunf = ΔH − TΔS). The main contributions to entropy are the conformational entropy of the polypeptide chain itself and ordering of water molecules around hydrophobic side chains of the protein. To elucidate the role of conformational entropy upon thermal unfolding in more detail, conformational dynamics in the time regime of picoseconds was investigated with neutron spectroscopy. Confined internal structural fluctuations were analyzed for α-amylase in the folded and the unfolded state as a function of temperature. A strong difference in structural fluctuations between the folded and the unfolded state was observed at 30°C, which increased even more with rising temperatures. A simple analytical model was used to quantify the differences of the conformational space explored by the observed protein dynamics for the folded and unfolded state. Conformational entropy changes, calculated on the basis of the applied model, show a significant increase upon heating. In contrast to indirect estimates, which proposed a temperature independent conformational entropy change, the measurements presented here, demonstrated that the conformational entropy change increases with rising temperature and therefore contributes to thermal unfolding.
